ABSTRACT
Plants are sensitive to photoperiods and are also equipped with systems to adjust their flowering time in response to various changes in the environment and developmental hormones. In the present study, previously generated rice OsWOX13 overexpression and newly generated OsWOX13 knockout lines constructed via CRISPR/Cas9 technology flowered 10 days earlier and 4-6 days later than the wild type, respectively. Quantitative real-time polymerase chain reaction analyses revealed that OsWOX13 might be involved in drought escape responses through the b-ZIP TRANSCRIPTION FACTOR 23 signaling pathway during rice flowering via photoperiod signaling genes such as Grain number, plant height and heading date 7, Early heading date 1, RICE FLOWERING LOCUS T1, Heading date 3a, and MADS14. Future investigations of OsWOX13 may provide insight into how plants adjust their flowering under stress conditions and how OsWOX13 could be precisely controlled to achieve maximum productivity in rice breeding.
OsWOX13 might be involved in rice flowering through a drought escape mechanism.
- Abbreviations
- DAS:
days after sowing
- NLC:
natural long-day conditions
- TF:
transcription factor
- WOX:
WUSCHEL homeobox transcription factor
After a certain period of vegetative growth, plants generate reproductive organs (Major 1980). Environmental factors such as day length (photoperiod) and temperature strongly influence this transition. The flowering network within plants is also associated with hormonal and stress response pathways to enable adaptation to harsh environments (Vicentini et al. 2023). Rice (Oryza sativa) is a facultative short-day (SD) plant whose flowering time is prolonged under long-day (LD) conditions, and the mechanisms underlying these photoperiodic responses are finely tuned (Zhou et al. 2021). Photoperiodic responses result in leaves producing florigen, which has been shown to be transported to the shoot apical meristem (SAM) and to induce floral meristem production (Tamaki et al. 2007). Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) are the main florigen-type mobile signals under SD and LD conditions, respectively (Kojima et al. 2002; Komiya et al. 2009).
In the model plant Arabidopsis, a facultative LD plant, the GIGANTEA (GI) protein promotes CONSTANS (CO) transcription in leaves, and the CO protein directly activates transcription of the florigen FLOWERING LOCUS T (FT). As a facultative SD plant, rice has evolved a mechanism similar to that of the Arabidopsis GI-CO-FT pathway but in the form of the unique OsGI-Hd1-Hd3a pathway (Corbesier et al. 2007; Salazar et al. 2009). Under SD conditions, Heading date 1 (Hd1) promotes heading and exhibits diurnal expression, with expression peaking at night (Yano et al. 2000; Hayama et al. 2003). Moreover, Hd3a is regulated by Early heading date 1 (Ehd1; Yano et al. 2000; Doi et al. 2004), and Ehd1 is regulated by activators or repressors (Kim et al. 2007; Ishikawa et al. 2011; Izawa et al. 2011). In contrast, Hd1 functions as an inhibitor under LD conditions (Yano et al. 2000). Ehd1 appears to be the main activator of Hd3a/RFT1 under LD conditions. Notably, the expression of Ehd1 is negatively regulated by Grain number, plant height and heading date 7 (Ghd7), a primary external coincidence regulator, PSEUDO-RESPONSE REGULATOR 37 (OsPPR37/Hd2/DTH7), and VERNALIZATION INSENSITIVE 3-LIKE 1 (OsVIL2; Xue et al. 2008; Ryu et al. 2009; Itoh et al. 2010; Wang et al. 2013; Yang et al. 2013). Recent studies have also suggested that the Hd1 and Ehd1 pathways are independent of florigen genes in rice under LD conditions. Days to heading on chromosome 2 (DTH2) is necessary for the expression of Hd3a and RFT1 and has a minor effect on the quantitative trait locus that promotes heading under LD conditions, suggesting the existence of a more finely tuned photoperiod-sensing mechanism (Wu et al. 2013).
Plants are sensitive to photoperiods and are also equipped with systems via which environmental factors, including abiotic stresses and hormones, influence flowering time. In particular, there are plant species that hasten flowering in response to mild drought stress as a form of drought escape (DE), as well as plant species that postpone flowering in response to drought conditions as a form of drought tolerance (DT; Riboni et al. 2013; Vicentini et al. 2023). b-ZIP TRANSCRIPTION FACTOR 23 (OsbZIP23) has been suggested to be a key player in the tolerance to drought and high-salinity stress and in the sensitivity to abscisic acid (ABA) in rice (Xiang et al. 2008). OsbZIP23 expression was also reported to be induced by low water treatment (LWT; Du et al. 2018), suggesting that OsbZIP23 may also play a positive role in DE through the negative regulation of Ghd7-Ehd1 signaling. Rice appears to have ABA-dependent and ABA-independent pathways involved in its DE response. Some light receptors, circadian components, and flowering-related genes, including PHYTOCHROME B (PhyB), Ghd7, and TIMING OF CAB EXPRESSION 1 (OsTOC1), have been found to be involved in the DE response in an ABA-dependent manner (Du et al. 2018). In contrast, several other flowering-related genes, including OsGI, EARLY FLOWERING 3-1 (OsELF3), OsPRR37, and MADS BOX GENE 50 (OsMADS50), were found to be involved in the regulation of DE independent of ABA. External stressors such as drought and ABA eventually converge at the level of transcriptional regulation of Ehd1, Hd3a, and RFT1, thus acting as integrators of multiple signals. Some transcription factors (TFs), such as the bZIP TF O. sativa ABA responsive element binding factor 1 (OsABF1) and its closest homolog, OsbZIP40, might be involved in DT responses rather than DE responses (Zhang et al. 2016). ABA, which is the major stress phytohormone, seems to have contrasting effects on flowering time in DE and DT responses, but it may help plants adapt to adverse conditions by enabling plants to adopt different strategies through the differentiation of developmental processes. ABA coordinates gene expression during plant adaptation to stress. Although ABA is considered a germination and root growth inhibitor that might help plants minimize water loss and expend less energy under stress conditions, in some cases, ABA promotes root growth and enables the root system to access water (Zhao et al. 2014). ETHYLENE RESPONSE FACTOR 33 (ERF33/OsDREB4-2/OS04g0549700) contains a potential nuclear localization signal, an AP2 DNA-binding domain and an ERF33 protein that is specifically bound to the drought-responsive element (Tian et al. 2005). Repetitive proline-rich proteins (RePRPs) are proline-rich glycoproteins with highly repetitive PX1PX2 motifs that exist only in monocotyledonous plants (Tseng et al. 2013). In rice, RePRPs play an essential role in the ABA/stress-mediated regulation of root growth and development. The RePRP2.2 (Os07g0418600) transcript is preferentially expressed in roots and panicles.
We previously reported that OsWOX13 is temporally regulated in vegetative organs and spatially regulated in flowers and seeds (Minh-Thu et al. 2018). OsWOX13 (Os01g0818400) was moderately upregulated in leaves and roots as early as 30 min after the onset of drought stress. The overexpression of OsWOX13 (OsWOX13-ov) under the control of the rap21 promoter in rice enabled survival despite drought, with flowering occurring early by 7-10 days, suggesting that early flowering is mediated by drought stress response TFs. In the present study, OsWOX13 knockout delayed heading by 4-6 days through the OsbZIP23, Ghd7, and Ehd1 rice flowering signaling pathways, suggesting that OsWOX13 mediates the abiotic stress response and flowering time via DE responses.
Materials and methods
Plant materials and growth conditions
The rice plants were grown in a greenhouse or in a paddy field at the National Institute of Agricultural Sciences in Jeonju, Korea (35.83 N, 127.05 E; Minh-Thu et al. 2018; Kim et al. 2021). The rice cultivar O. sativa ssp. Japonica cv. Ilmi was used as a wild type (WT) for the generation of the overexpression and knockout lines. Wild-type Ilme (WT-IM) (WT) is a late-flowering japonica cultivar that headed on August 16-17 (101-2 days after sowing [DAS]). Seeds were germinated in a greenhouse on May 8th and transplanted in paddy fields. Phenotypic observations and measurements were performed on plants grown in the paddy fields. A total of 15-20 plants were observed for heading in paddy fields. Photos were taken after the plants were transported to the pots.
oswox13 knockout lines generated via CRISPR/Cas9 vector construction
The sgRNA vector contained BsaI between the OsU3 promoter and the sgRNA and attachment site for the attL and attR recombination in a gateway (LR) reaction (Ma et al. 2015). To generate the construct expressing sgRNA, we designed 2 oligomers: forward (5′-ggcaGACAAGGCCAAGGCGTCCTC-3′) and reverse (5′-aaacGAGGACGCCTTGGCCTTGTC-3′) oligomers. Two oligo strands were annealed in equimolar amounts (100 µm). The annealed oligomers were ligated to an sgRNA vector predigested with BsaI. In the LR reaction, OsU3-sgRNA was cloned and inserted into the pOsCas9-HYG vector carrying the Cas9 and hygromycin genes under the control of the ubiquitin and CaMV 35S promoters, respectively. The construct was sequenced and subsequently transformed into Agrobacterium tumefaciens LBA4404 cells via standard molecular biological techniques.
Direct DNA amplification for polymerase chain reaction and sequencing
Genomic DNA from transformed and WT plants was isolated via the NaOH extraction method (Werner et al., 2002). Polymerase chain reaction (PCR) analysis was carried out to analyze the insertion/deletion (indel) of a given target gene via gene-specific primers (Table S1). Each primer pair was designed via the Primer-BLAST tool (http://ncbi.nlm.nih.gov). PCR analysis was also performed to detect the presence of T-DNA via Cas9 gene-specific primers. The PCR reagents were purchased from SolGent (Republic of Korea), and the reactions were performed with a 2720 Thermal Cycler (Applied Biosystems, USA). Each PCR product was loaded and separated on a 2% agarose gel and subsequently purified via a HiYield Gel/PCR DNA Mini Kit (RBC, Taiwan) according to the manufacturer's instructions. The PCR products were sequenced via the Sanger sequencing method via a gene-specific primer, and target indels caused by Cas9 cleavage were analyzed from an ABI chromatogram file. Nucleotide sequences were compared to those of OsWOX13 in the Rice Annotation Project (RAP) database. Deduced sequences were handled with BioEdit (https://bioedit.software.informer.com).
RNA extraction, cDNA synthesis, and quantitative real-time PCR analysis
Total RNA was extracted from single tillers of WT and transgenic rice plants via a Hybrid-R system (GeneAll, Republic of Korea) at approximately 10 a.m. at 84, 42, 28, and 20 days before heading (August 16th or 17th) and at 17 (May 25th), 59 (July 6th), 73 (July 20th), and 81 (July 28th) DAS. Afterward, 1 µg of total RNA was used as a template for reverse transcriptase reactions via a RevertAid H Minus First-Strand cDNA Synthesis Kit (Fermentas, USA) according to the manufacturer's instructions. Gene-specific primers were designed via the Primer-BLAST tool (Table S1. PCR was performed on a StepOne real-time PCR detection system (Applied Biosystems, USA) according to the manufacturer's instructions. The data were normalized to the expression of ubiquitin 5. Changes in gene expression were analyzed with StepOne software (Applied Biosystems, USA). Quantitative real-time PCR (qRT-PCR) was carried out for 3 biological replicates. Expression was assessed by evaluating threshold cycle (CT) values. The relative expression levels of the tested genes were normalized to that of the ubiquitin gene and calculated via the 2−ΔΔCT method (Livak and Schmittgen, 2001). The data were summarized with the Perl script (https://www.perl.org), and 1-way analysis of variance (ANOVA) and significance tests were performed with the DunnettTest function in the DescTools library in R statistical language (https://cran.r-project.org/).
RNA sequencing and data processing
Total RNA was purified via a TruSeq RNA Sample Preparation Kit (Illumina, USA) as described previously (Kim et al. 2021). The fragmented cDNA was then purified, amplified via PCR, and sequenced with a NovaSeq 6000 device. For each sample, 6.0-8.0 Gb (64-88 × 106 paired-end reads) of data were generated. The raw sequence reads were mapped to the rice genome sequence (IRGSP-1.0_genome) in the RAP database (http://rapdb.lab.nig.ac.jp). Differential exon usage was tested with the DEXSeq and limma packages in Bioconductor (bioconductor.org). The median count was adjusted by adding 1 to avoid trivial division errors. Gene Ontology (GO) (https://geneontology.org/) and enrichment analyses were performed with Pantherdb (https://pantherdb.org/) via pthr_go_annots.py (https://github.com/pantherdb/pantherapi-pyclient). The adjusted P values of the GO enrichment data were scaled from 0 to 5 or −5 for the up- and down-regulated genes, respectively, as shown in Table S3. For further analysis of significant transcripts from RNA-sequencing (RNA-seq) counts and GO terms, distance and cluster were calculated with the dist function associated with the Euclidean method, and hierarchical clustering was performed with the hclust function with parameters, with an average based on the results obtained by R. The enriched downregulated and upregulated genes are color coded with green and red, respectively, in heatmap.2.
Results and discussion
Vector construction for knocking out the OsWOX13 gene via CRISPR/Cas9-based technology
For subsequent analysis, we constructed a CRISPR/Cas9-based vector to knock out the OsWOX13 gene (Naito et al. 2015). A 20-nt target sequence from exon I of OsWOX13 was chosen and constructed as described in the “Materials and methods” section (Figure 1a). A total of 17 T0 mutant lines were generated via Agrobacterium-mediated transformation (oswox13 knockout [oswox13-ko]), and PCR amplification of the target regions suggested that 3 and 14 lines contained homozygous and heterozygous mutations, respectively. The 3 homozygous lines were designated oswox13-ko10, oswox13-ko2, and oswox13-ko7 (Figure 1b). Among these homozygous lines, oswox13-ko10 and oswox13-ko2 contained T and A insertions in the first exon of OsWOX13, respectively, whereas oswox13-ko7 contained 2 deletions. These insertions and deletions introduced frame shifts and premature stop codons (Figure S1). We also tracked the segregation of the transgene (T-DNA) in the T1 population of the T1 mutants via a PCR assay of Cas9, which is an element of T-DNA (Figure S2). Among the 10 T1 oswox13-ko10 progeny plants, 4 had negative Cas9 PCR results, indicating a transgene-free homozygous mutant. However, 10 T1 oswox13-ko2 events involved the Cas9 gene. Two T1 oswox13-ko7 progeny plants produced a transgene-free homozygous mutant.
The OsWOX13 gene was knocked out via CRISPR/Cas9 technology, and the resulting oswox13-ko lines exhibited delayed flowering under natural LD conditions. (a) KO of OsWOX13. Two oligo strands for the target site were annealed and ligated to the sgRNA vector digested with BsaI (Ma et al. 2015). (b) Among the 17 T0 mutant lines (oswox13-ko), 3 and 14 lines were homozygous and heterozygous, respectively. The three homozygous lines were designated oswox13-ko10, oswox13-ko2, and oswox13-ko7. (c) WT-IM (WT) showed heading at 101-102 days (August 17th) under natural LD conditions (NLC) and is referenced at 101 days as 0 on the x-axis. The numbers of plants from the OsWOX13-ov8, ov1, and ov30 lines at the heading stage (Figure 1c top) at approximately 94.6, 96.4, and 96.8 days after sowing (DAS; Minh-Thu et al. 2018), respectively, are shown in a bar graph. (−) represents the days ahead of WT heading. In contrast, the heading of the oswox13-ko10, oswox13-ko2, and oswox13-ko7 lines (T2 generation) occurred at DAS 105.8 DAS (August 21st), 106.1 DAS (August 22nd), and 106.8 DAS (August 23rd), respectively, and these represented delays of 4.3, 4.7, and 5.4 days, respectively (Figure 1c bottom). Fifteen to twenty plants were observed for heading in paddy fields. (d) Rice plants grown in paddy fields were transferred to pots at 101 (left panel), 107 (middle panel), and 120 (right panel) DAS, and photos were taken.
oswox13-ko plants exhibited a 4-6-day delay in flowering in paddy fields
Cas vector-free oswox13-ko10-6 (ko10) and oswox13-ko7-1 (ko7) were chosen for further analysis. oswox13-ko2-1 (ko2) was also chosen, although it contained a Cas vector. The heading date of the T2 oswox13-ko lines was examined. In Jeonju, Korea (35.83 N, 127.05 E), WT-IM (WT) headed at approximately 101.5 (s.d. 2.1) DAS (August 17th) in the paddy field, whereas OsWOX13-ov8, ov1, and ov30 lines at the heading stage (T9 generation; Figure 1c top) headed at approximately 94.6 (August 10th), 96.4, and 96.8 DAS, respectively (Minh-Thu et al. 2018). A 1-tailed t-test revealed that all the P values were less than 0.01, suggesting that the differences in heading dates were significant. In contrast, the oswox13-ko10, oswox13-ko2, and oswox13-ko7 lines headed at 105.8, 106.1, and 106.8 DAS, respectively. A 1-tailed t-test revealed that all the P values were less than 0.01, suggesting that the differences in heading dates were significant. Thus, the phenotypes of the oswox13-ko10, oswox13-ko2, and oswox13-ko7 progeny lines were characterized by delays in flowering of 4.3, 4.7, and 5.4 days, respectively, compared with the flowering time of the WT (Figure 1c). Statistical analysis revealed that these differences in heading duration were significant, with P values <0.01. At 101 DAS, the WT plants exhibited heading, and the OsWOX13-ov8 plants had fully headed (Figure 1d, left panel). At 107 DAS, the WT plants and OsWOX13-ov8 plants had fully headed, and the KO lines had started to head (Figure 1d, middle panel). Thus, compared with the heading of the WT plants, the heading of the KO lines was moderately delayed, by 4-6 days.
RNA-seq analysis revealed that ABA-responsive genes and flowering genes were modulated by overexpression and knockout of OsWOX13
WT-IM is a late-flowering japonica cultivar that headed on August 16-17 (101-2 DAS) in paddy fields and is considered a natural LD cultivar under natural LD conditions. The expression of florigens such as Hd3a and RFT1 reportedly increases 30-35 days (70 DAS) before heading, when the expression of MADS-box transcription factor 14 (OsMADS14) and OsMADS15, which are downstream of Hd3a and RFT1, increases in the inflorescence meristem (IM) starting from the primary panicle branch generation stage (Komiya et al. 2008, 2009). To examine how the expression of ABA/stress- and florigen-related genes is modulated by OsWOX13 throughout this period, WT, OsWOX13-ov8, and oswox13-ko10 plants were sampled at 10 a.m. at 59, 73, and 81 DAS. As a reference, the WT at 17 DAS was also sampled. Total RNA was collected from a whole tiller above ground and sequenced as described in the “Materials and methods” section. All the counts were mapped to the IRGSP-1.0_genome in the RAP database (http://rapdb.lab.nig.ac.jp). After all the counts were normalized, the counts were referenced at 17 DAS. This yielded 52 817 transcripts (Table S2) and revealed that approximately 5900 transcripts were modulated by that time point, with changes below the threshold values of 1.0 for log2-fold change and 0.05 for the adjusted P value. The expression of the genes gradually changed from 59 DAS to 73 DAS and drastically changed at 81 DAS, suggesting that the reproduction stage had begun (Figure S3). Several enriched GO terms were observed among these lines: GO:0045454_cell redox homeostasis, GO:0009414_response to water deprivation, GO:0060862_negative regulation of floral organ abscission, GO:0010115_regulation of ABA biosynthetic process, GO:0009873_ethylene-activated signaling pathway, and GO:0062211_root regeneration (Table S3 and Figure 2). GO:0009750_response to fructose, GO:0080148_negative regulation of response to water deprivation, and GO:0080168_ ABA transport were enriched in oswox13-ov8, and GO:0007623_circadian rhythm was enriched in oswox13-ko10, implying that OsWOX13 regulates ABA transport, circadian rhythm, etc. At 73 DAS, the enriched GO terms were GO:0009416_response to light stimulus, GO:0009751_response to salicylic acid, and GO:0009658_chloroplast organization. Interestingly, GO:0009911_positive regulation of flower development was enriched in OsWOX13-ov8, suggesting that floral morphogenesis had begun. At 81 DAS, GO:0009911_positive regulation of flower development, GO:0030422_production of siRNAs involved in post-transcriptional gene silencing by RNA, GO:0030244_cellulose biosynthetic process, and GO:0009833_plant-type primary cell wall biogenesis were enriched in all the lines.
Enriched GO terms. GO enrichment analysis of the genes was performed as described in the “Materials and methods” section. D: The enriched terms for downregulated genes are underlined, and the genes are color coded in green. U: The enriched terms for upregulated genes are shown, and the genes are color coded in red. The adjusted P values for the GO enrichment analysis are color coded as red and green for the up- and downregulated genes, respectively (scaled enrichment).
ABA-responsive genes were modulated, but ABA core signaling genes did not respond much
To examine whether OsWOX13 modulates ABA or stress responses, we tested the expression of genes whose expression was modulated by ABA/abiotic stress. At 59 DAS, OsWOX13 was expression in the WT was much lower under stress than that in the control (Figure 3, OsWOX13). In contrast, its expression level decreased 1.6-fold in ko10, and this difference was significant according to Dunnett's test, as described in the “Materials and methods” section. At 73 DAS, OsWOX13 expression slightly increased in WT and ov8, whereas that in ko10 remained low up to 81 DAS. The gene introduced into ov8 (Minh-Thu et al. 2018) was examined with an OsWOX13 gene-specific forward primer, OsWOX13F, and a reverse primer, attB2, designed for the gateway system in the overexpression vector (Table S1). This gene was highly expressed at 59 and 73 DAS (Figure 3, OsWOX13F/attB2), suggesting that the OsWOX13 overexpression vector was effective.
qRT-PCR analysis of ABA/stress-related transcripts. ERF33 (OsDREB4-2/OS04g0549700) and RePRPs play essential roles in ABA/stress-mediated regulation of root growth and development. DTH, days to heading of WT. WT is a late-flowering japonica cultivar that exhibited heading on August 16-17 (101-2 DAS), and the DTH was calculated in reverse. The dates corresponding to DAS and DTH are indicated below. The expression of these genes relative to that of UBQ5 is presented. Three biological replicates were performed. Dunnett’s test was performed to determine the significance of the differences, with the result for the WT at the same DAS used as a control. Dunnett's test P values are indicated as follows: 0; “***”, 0.001; “**”, 0.01; “*”, 0.05.
To test whether the expression of OsWOX13 is modulated by ABA/stress, we tested several genes that are modulated by abiotic stress. The RNA-seq results suggested that ERF expression was greater in OsWOX13-ov8 (1.8-fold) than in the WT (1.3-fold) and oswox13-ko10 (1.2-fold) at 59 DAS, whereas that in the WT was slightly greater than that in ov8 at 73 DAS (Table S2). The expression of these genes increased greatly (2.8-4.8-fold) at 81 DAS. Indeed, qRT‒PCR also revealed that the expression of ERFs was modulated during these periods, although the pattern of expression was slightly different from that determined via RNA-seq. At 59 DAS, ERF33 was expressed at relatively high levels in the OsWOX13-ov8 plants. At 73 DAS, ERF33 expression was greater than it was at 59 DAS, and among the different lines, the oswox13-ko10 line presented the highest ERF33 expression (Figure 3 ERF33). However, as OsWOX13 seemed to regulate the expression levels of EFR33 in opposite directions between 59 and 73 DAS, its effects remain to be clarified.
We also monitored RePRP2.2, which is reported to be expressed in roots and panicles. The RNA-seq counts suggested that expression was strongly suppressed from 59 to 73 DAS (Table S2), and the degree of suppression in WT-IM decreased almost 30-fold, whereas it decreased 10.1- and 1.5-fold in the ov8 and ko10 lines, respectively. Interestingly, the expression of these genes increased approximately 2.2-fold at DAS81, suggesting that their expression pattern drastically changed upon entry into reproduction stages. qRT-PCR also revealed that these repression patterns occurred from 59 to 73 DAS, and RePRP2.2 was expressed at relatively high levels at 59 and 73 DAS in the oswox13-ko10 and OsWOX13-ov8 rice plants, respectively (Figure 3, RePRP2.2). However, gene expression in both the overexpression and knockout lines was greater than that in the WT. These data suggest that the regulation of these genes could be more complex.
We also tested several of the core ABA signaling genes (Kim et al. 2012; Lynch et al. 2012). OsPYL/RCAR5, SAPK2, and ABI5 were not strongly modulated at 59 and 73 DAS, suggesting that OsWOX13 is likely independent of or downstream of the signaling systems to which these genes belong (Figure S4). In addition, qRT-PCR analyses revealed that ABA/stress-related genes such as ERF33 and RePRP2.2 were involved in the phenotypes in an ABA core signaling-independent manner.
OsWOX13 might participate upstream of OsbZIP23 in DE through negative regulation of Ghd7-Ehd1 signaling in the natural LD response of rice
In the flowering pathway, especially under natural LD conditions, Ghd7, Ehd1, RFT1, Hd3a, and MADS14 are important components (Zhou et al. 2021; Vicentini et al. 2023). OsbZIP23 expression was reported to be induced by LWT (Du et al. 2018), suggesting that OsbZIP23 may also play a positive role in DE through the negative regulation of Ghd7-Ehd1 signaling. OsbZIP23 expression was also found to be induced 3.3- and 2.7-fold in leaves and roots, respectively, within 2 h of drought treatment (transcript evidence AK072062 in Table S2 in Minh-Thu et al. 2013). At 59 DAS, OsbZIP23 expression was marginally greater in the OsWOX13-ov8 plants than in the WT-IM plants. However, OsbZIP23 expression was repressed nearly 2-fold in the oswox13-ko10 plants, suggesting that it might positively regulate flowering induction and that OsWOX13 functions as an upstream regulator of OsbZIP23 (Figure 4). OsbZIP23 has been proposed to be a negative regulator of Ghd7 in the DE response in rice. Ghd7, which encodes a CCT domain protein, has major effects on numerous traits in rice, including the number of grains per panicle, plant height, and heading date (Xue et al. 2008; Du et al. 2018). Ghd7 is upstream of Ehd1 and Hd3a in the LD flowering pathway and functions as a negative regulator. Ghd7 and Ehd1 have been shown to be critical components of a mechanism via which slight differences in day length (usually 13.5 h) lead to the induction of Hd3a transcription (Itoh et al. 2010). Ghd7 and Ehd1 are floral repressors and promoters, respectively, under LD conditions. In the present study, Ghd7 expression at 59 DAS decreased 2.7-fold in OsWOX13-ov8 but increased in oswox13-ko10 plants (Figure 4, Ghd7). In contrast, Ehd1 expression was induced 30.8-, 66.6-, and 11.9-fold in the WT, OsWOX13-ov8, and oswox13-ko10 plants, respectively, with clear induction of the flowering pathway (Figure 4, Ehd1). The fold increase in the expression of genes related to this signaling pathway seemed to increase, as previously reported (Xue et al. 2008).
qRT-PCR analysis of the photoperiod-related genes Ghd7, Ehd1, RFT1, Hd3a, and MADS14. RFT1 and Hd3a are florigens. The APETALA1 (AP1)/FRUITFULL (FUL)-like gene MADS14 specifies the identity of the IM downstream of the florigen signal. The dates corresponding to DAS and DTH are indicated below. The expression of these genes relative to that of UBQ5 is presented. Three biological replicates were performed. Dunnett’s test was performed to determine the significance of the differences, with the result for WT-IM at the same DAS used as a control. Dunnett's test P values are indicated as follows: 0; “***”, 0.001; “**”, 0.01; “*”, 0.05.
Florigens such as RFT1 and Hd3a are expressed in the phloem of rice leaf blades and move to the SAM to initiate floral transition and downstream Ghd7 and Ehd1 signaling pathways during LD flowering of rice (Kojima et al. 2002; Komiya et al. 2009). Hd3a, in association with a 14-3-3 protein in the cytoplasm, translocates to the nucleus and forms the florigen activation complex (Tsuji et al. 2013). This complex regulates the APETALA1 (AP1)/FRUITFULL (FUL)-like genes MADS14, MADS15, and MADS18. MADS14 appears to act in conjunction with the meristem to specify the identity of the IM downstream of the florigen signal. We tested RFT1, Hd3a, and MADS14 induction in WT-IM, OsWOX13-ov8, and oswox13-ko1 plants. RFT1 is the closest homolog to Hd3a and is a major floral activator under LD conditions (Komiya et al. 2009). RFT1 expression in the OsWOX13-ov8 plants began to be induced at 59 DAS and peaked at 73 DAS, while that in the WT-IM and oswox13-ko10 plants peaked at 73 DAS, but the degree of induction was lower than that in the OsWOX13-ov8 plants. After 2 weeks, Hd3a expression increased 7-, 30-, and 15-fold in the WT, OsWOX13-ov8, and oswox13-ko10 plants, respectively, compared with that in the WT plants at 59 DAS. At 73 DAS, Hd3a expression in the OsWOX13-ov8 plants was 2-fold greater than that in the oswox13-ko10 plants. Moreover, MADS14 expression at 73 DAS increased 20-, 70-, and 8-fold in the WT, OsWOX13-ov8, and oswox13-ko10 plants, respectively, compared with that at 59 DAS.
At 81 DAS, the expression of MADS14 increased 3.5-fold and 2.5-fold in the overexpression and knockout lines, respectively, compared with that in the WT. These results indicate that knockout of the OsWOX13 gene repressed rice flowering by downregulating the expression of MADS14.
qRT-PCR analyses suggested that OsWOX13 might be involved in DE responses through OsbZIP23, Ghd7, Ehd1, and Hd3a in the rice flowering signaling pathway, although the flowering times of the mutants and overexpression lines under LWT conditions have not been investigated.
The number of grains per panicle of some of the ov lines was greater, and the panicle lengths of the ko lines were shorter, than those of the WT lines
The appearances of the seeds of the WT, OsWOX13-ov (T9), and oswox13-ko (T3) lines were not very different (Figure S5). The average grain lengths of the WT and OsWOX13-ov lines were 0.74 and 0.72 cm, respectively, and those of the oswox13-ko lines were 0.76 cm. The 250-grain weights of the WT, OsWOX13-ov, and oswox13-ko lines were not very different. Yield-related factors were also tested (Figure 5). The number of grains per panicle in the ov1 and ov8 lines (180-190) was greater than that in the WT (130) plants, with P values ranging from 0.05 to 0.1 (Figure 5a). In contrast, the number of grains of the KO lines was lower than that of the WT plants. The heights of the plants of the overexpression and KO lines were not very different from those of the WT plants. However, the oswox13-ko10 plants were taller than the WT plants. The panicle lengths of the plants of the overexpression lines were not very different, whereas the panicle lengths of the plants of the KO lines were consistently shorter than those of the WT plants (Figure 5b).
Yield factors, such as grains per panicle (a) and panicle length (b). Measurements (3-5 plants) of the number of panicles in the OsWOX13 overexpression and knockout lines and in the WT. A 1-tailed t-test was performed. *P < 0.05 and **P < 0.01 indicate significant differences.
It has been proposed that plants cope with drought conditions either by hastening flowering (DE) or by postponing flowering (DT; Riboni et al. 2013). In a previous study, PhyB, OsbZIP23, Ghd7, and OsTOC1 were found to be involved in an ABA-dependent manner during the DE response, whereas OsGI, OsELF3, OsPRR37, and OsMADS50 were involved in an ABA-independent manner (Du et al. 2018). The stressors that induce DE eventually converge at the level of transcriptional regulation of Ehd1, Hd3a, and RFT1, thus acting as integrators of multiple signals. In our study, OsbZIP23, Ghd7, Ehd1, RFT1, Hd3a, and MADS14 were also modulated. The use of OsWOX13 might provide insights into how plants adjust flowering under stress conditions and may allow precise control for maximum productivity. In addition, the number of grains increased for some of the lines (Figure 5), and the panicles of the KO lines seemed to be shorter than those of the WT lines (Figure 5).
In the northern temperate zone, rice cultivation is restricted by the onset of winter, yet sufficient vegetative growth must occur for maximum yield. Most rice cultivars in these areas have a heading date of approximately 100 days and were chosen for cultivation in the present study. A geographical survey of rice cultivar distribution suggested that the Hd1, Ehd1, and Ghd7 genes are the main targets in rice breeding (Izawa 2007), and weak Hd1, Ehd1, and Ghd7 alleles have been used for the greatest productivity in northeastern Asia. The major genes involved in photoperiod sensitivity strongly affect flowering. Hd1 mutation in the Kasalth cultivar resulted in flowering occurring 10 days earlier than that in the Nipponbare cultivar under LD conditions. Nonfunctional Ehd1 and RFT1 RNAi plants flowered 80-100 days later than did the WT under LD conditions (Lin et al 2000; Doi et al. 2004; Komiya et al 2009). Only a few flowering time genes that function in adaptation have been identified, and their underlying molecular mechanisms have been elucidated. DTH2 was found to exhibit a 7.4-day delay in heading date under natural LD conditions and seems to be suitable for Northeast Asia (Wu et al 2013). In light of these findings, flowering that occurs several days early, as observed in the overexpression lines in our previous study, and delayed flowering, as observed with OsWOX13 knockout in the present study, may provide opportunities not only for understanding DE mechanisms but also for producing suitable cultivars for breeding efforts.
Data availability
The RNA-seq data are available at NCBI SRA under BioProject: PRJNA1113822.
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (YKK; Grant No. RS-2023-00241813).
Disclosure statement
No potential conflict of interest was reported by the authors.
Acknowledgements
The author thanks Drs. Joung Sug Kim and Kyong Mi Jun for performing qRT-PCR. Dr. Gang-Seob Lee documented the phenotypes of the rice lines in the field.
